Cockayne Syndrome (CS) is an autosomal recessive disorder, characterized by growth failure, neurological abnormalities, premature aging symptoms, and cutaneous photosensitivity, but no increased cancer incidence. CS is divided into two complementation groups: CSA (mutation in CKN1) and CSB (mutation in ERCC6). Of the patients suffering from CS, 80% have mutations in the CSB gene. Emerging evidence indicates a role for CSB in facilitating the BER response. For instance, we recently identified a novel physical and functional interaction between CSB and the major human abasic endonuclease, APE1, which operates centrally in the BER pathway. However, the precise molecular contributions of CSB to this repair process remain poorly defined. Furthermore, although CSB harbors the seven conserved ATPase motifs found in the SWI2/SNF2 helicase-like superfamily of chromatin remodeling proteins, its biochemical properties remain largely uncharacterized. We have therefore taken on a major effort to delineate the biochemical functions of CSB. Our recent work has revealed that Ca2+ is a novel metal cofactor for CSB catalyzed DNA-dependent ATP hydrolysis, but that CSB lacks detectable ATP dependent helicase and single- or double-stranded nucleic acid translocase activities in the presence of either Ca2+ or Mg2+. In addition, we have discovered that (i) CSB supports ATP independent complementary strand annealing of not only DNA/DNA duplexes, but DNA/RNA and RNA/RNA duplexes, with Ca2+ again promoting optimal activity;(ii) CSB forms a stable protein:DNA complex with a 34mer double-stranded DNA in electrophoretic mobility shift assays, independent of divalent metal or nucleotide (e.g. ATP);and (iii) CSB stably binds a range of nucleic acid substrates, including bubble and pseudo-triplex double-stranded DNAs that resemble replication and transcription intermediates, as well as forked duplexes of DNA/DNA, DNA/RNA, and RNA/RNA composition, the latter two of which do not promote CSB ATPase activity. Moreover, association of CSB with DNA, independent of ATP binding or hydrolysis, was found to displace or rearrange a stable pre-bound protein:DNA complex, a property likely important for its roles in transcription and DNA repair. Future work, in collaboration with Dr. Vilhelm Bohr, will continue to examine the in vitro activities of CSB on key DNA and RNA transaction intermediates, and will elucidate the contributions of the unique N- and C-terminal portions of the protein that likely impart functional specificity. The hypothesis is that CSB operates as an auxiliary protein in BER by inducing topological changes in nucleic acid targets and directing specific protein interactions, and that these functions are critical to the manifestation of the inherent disease pathologies associated with CS patients. XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSBR). Recent data has found an association of XRCC1 with proteins causally linked to human spinocerebellar ataxias - aprataxin and tyrosyl-DNA phosphodiesterase 1 - implicating SSBR in protection against neuronal cell loss and neurodegenerative disease. We demonstrated that shRNA lentiviral-mediated XRCC1 knockdown in human SH-SY5Y neuroblastoma cells results in a largely selective increase in sensitivity of the nondividing (i.e. terminally differentiated) cell population to the oxidizing agents, menadione and paraquat. Primary XRCC1 heterozygous mouse cerebellar granule cells were found to exhibit increased strand break accumulation and reduced survival due to increased apoptosis following menadione treatment. Moreover, knockdown of XRCC1 in primary human fetal brain neurons resulted in an enhanced sensitivity to menadione, as indicated by increased levels of DNA strand breaks relative to control cells. The cumulative results implicate XRCC1, and more broadly SSBR, in the protection of nondividing neuronal cells from the genotoxic consequences of oxidative stress, and suggest that a set of ataxia patients may exist that arise from genetic defects in XRCC1. We are currently assessing the role of this protein in age-related pathologies using heterozygous mice, and will concomitantly evaluate the effect of XRCC1 haploinsufficiency on neurodegeneration and cancer proneness following defined insults.